The present invention relates to a cassette for use in the formation of an electrophoretic gel. More particularly, the invention relates to such cassettes made of two parts and having locking means for holding the two parts together in a desired orientation. The subject invention also relates to a method for separating analytes, such as bio-organic molecules by the use of the above-mentioned cassettes.
Electrophoresis is the resolution of a complex mixture of macromolecules on the basis of charge and/or size under the influence of an electric field. Usually, separation of the charged molecules is based on the strength of the electrical field and the net charge, size and shape of the molecules. The separation can also, at least in part, be affected by other parameters, such as isoelectric points, ionic strength, viscosity and temperature of the medium in which the charged molecules are moving. As proteins and other biological molecules, such as DNA, RNA, enzymes, carbohydrates and the like are charged, electrophoresis techniques are ideal to separate them for analytical or preparative purposes.
It is common practice to use a cassette for conducting gel electrophoresis. The cassette comprises two flat plates, usually transparent glass or plastic, separated by spacing means and sealed along the sides and bottom edges to provide a void or confined space between the plates for placement of a suitable separation gel medium or electrophoresis gel such as agarose or a polyacrylamide in liquid form and its subsequent gelling or polymerization. The bottom of the cassette may be sealed with tape, another spacer, or by compression against a gasket. Once sealed on the edges and bottom, a solution of acrylamide, polymerization catalysts and buffers are introduced into the cassette and allowed to polymerize. After the gel has formed, the seal on the bottom, if used, is removed to provide an electrical contact for electrophoresis.
Automating this casting procedure is a means of reducing cost, user labor, and improving reproducibility. When a separation is run on the gel in the cassette, the top portion of the gel is in contact with a buffered solution and the lower portion of the gel is in contact with a second buffered solution. An electric current is applied to the buffered solution(s) or the gel, causing the migration and separation of the samples. By convention, the samples, for example, negatively charged proteins and nucleic acids, tend to run from the cathode (the top of the cassette, where the sample is placed) to the anode of the gel.
For the purposes of describing this invention, reference to the top or upper end of a cassette means the end of the cassette at which the wells are formed and in the instance of negatively charged samples, the cathode end of the gel. Additionally, the use of the terms “gelled” and “gel” refers to a solid state of the electrophoretic medium whether it be due to the cooling of an agarose gel or the polymerization of an acrylamide or acrylamide derivative.
In protein or nucleic acid fragment analysis, it is desirable to run more than one sample in a gel and to keep the samples separate. This is accomplished by sample separating means which is most commonly provided by the formation of wells at the sample-loading end of the gel (ie near the upper end of the cassette). Sample wells are formed usually by means of a removable piece or “comb” with teeth having the desired shape of the wells and having straight (level) substantially perpendicular ends to the teeth. The comb is brought into contact with the liquid separation medium before it gels or polymerizes and is positioned so that the teeth extend into the liquid medium while it gels. After the gel has set, the comb is removed to leave wells where samples can be positioned.
To improve the separation of certain types of molecules, it is common practice to change the composition of the gel medium in the direction of the electrophoresis. This change in composition can, for example, take the form of a discontinuity formed by a stacking gel at the upper most portion of the gel and a resolving gel at the lower most portion of the gel. Another example of a change in composition is the commonly used gradient gel consisting of a continuous gradient or a discreet gradient of bands each of uniform but different composition or any combination of continuous and discreet gradient. In order to provide accurate sample resolution, it is necessary that the desired electrophoresis composition remain undisturbed until the gel medium has gelled or polymerized completely, particularly in the instance of stacking and/or gradient gels.
Additionally, the uniformity of the gel is important to the quality of the separation obtained, for example, uniformity in the porosity of the gel or regions of the gel, uniformity (level and perpendicular to the plates) in the edge of the gel which first contacts the samples, uniformity in the thickness of the gel and uniformity (level and perpendicular to the plates) in the interface between discreet bands of gels (for example, between gradient gels or between stacking and resolving gels). Thus, it is desirable that the liquid gel-forming medium introduced into the cassette be disturbed as little as possible while gelling.
Casting polyacrylamide gels for vertical electrophoresis is commonly done by creating a cassette formed of two glass or plastic parallel sheets. Typically, the parallel sheets are held in position by a pair of spacers located at the vertical edges of the sheets. These spacers typically perform the functions of connecting the parallel sheets and keeping the faces of the sheets apart, creating a cavity for receiving the electrophoretic gel. The spacers also serve to maintain the sheets in substantially parallel orientation. The connection between the parallel sheets is sometimes effected by the use of corresponding ridges and recesses which form a friction-fit or snap-fit. This means of connecting the parallel sheets has been found to be inadequate. For instance, the connection may not be entirely even along the lengths of the spacers and small gaps may exist at points between the corresponding ridges and recesses. These gaps can cause slight variations in the distance between the parallel sheets. The gaps can also cause leakage of the gel to occur. Another problem with this means of connecting the parallel sheets is that there is nothing to prevent the sheets from undergoing some movement relative to one another. For instance, if the cassette is inadvertently knocked or bumped (perhaps during transportation), the connection between the sheets may be loosened and they may separate slightly from one another. In this case, the space between sheets may not be entirely uniform throughout.
In order to inhibit leakage and to enhance the connection between the sheets, the spacers of one wall member are sometimes glued or welded to the other wall member. However, the use of glue or welding is still not sufficient to ensure that the faces of the parallel sheets are in fact completely parallel over their entire areas. This is often because the spacers may not correctly engage the opposite wall member at all points along the spacers. For instance, small gaps may still exist at some points. Similarly, the use of glue or welding may cause slight inconsistencies resulting in non-uniform engagement between the spacers and the opposite wall members. When the opposed wall members are not completely parallel, the electrophoretic gel will not be of uniform thickness. The nearer the gel can be to having uniform thickness throughout, the better and more reliable will be the results of the electrophoretic procedure.
Accordingly, the present invention is directed towards a cassette for holding an electrophoretic gel having improved uniformity of thickness of the gel.